Recombinant DNA technology has enabled the utilization of microbial organisms for the controlled production of various useful heterologous protein products. Gram positive bacteria, such as Bacillus subtilis, are often used as host organisms to produce useful protein products and the bacteria are engineered to extracellularly secrete the heterologous protein products directly into the growth medium. The protein products are then recovered from the growth medium.
During post-exponential growth, wild type Bacillus subtilis bacteria secrete, among other enzymes, several lytic proteins called proteases that degrade foreign or heterologous proteins in the growth medium. Due to the action of the proteases, it was often impossible to obtain large quantities of intact heterologous proteins secreted from B. subtilis, especially if they are of eukaryotic origin. The inactivation of the major extracellular protease genes encoding neutral (npr) and alkaline protease (apr) reduced the level of extracellular protease activity considerably (See, Kawamura F., and R. H. Doi (1984) "Construction of a Bacillus Subtilis double mutant deficient in extracellular alkaline and neutral protease." J. Bacteriol: 160: 442-444.), but a residual level was left. Depending on the sensitivity of the proteins to be produced the remaining proteases can still cause degradation. Therefore, the characterization of one of the remaining proteases, in particular purified serine protease, Epr, and isolated DNA sequences encoding such a serine protease would facilitate the construction of a triple extracellular protease, aprE, nprE and epr, deficient Bacillus subtilis strain. The use of this strain should increase the yield and stability of secreted heterologous protein products.